Phragmoplastin and methods of examining cell plate development

ABSTRACT

The present invention provides new tools and methods for identifying herbicides and potential herbicides which affect cell plate formation and development. A new protein, phragmoplastin, has been discovered which is associated with cell plate membrane vesicles during cytokinesis. By visualizing the phragmoplastin, one is able to examine the development of the cell plate particularly in response to herbicides or potential herbicides. One method of visualizing phragmoplastin employs immunocytochemical techniques with anti-phragmoplastin antibodies. Another method of visualizing phragmoplastin employs cells which are transformed with a DNA molecule which encodes a chimeric phragmoplastin protein comprised of phragmoplastin and a luminescent tag or protein, fused to the phragmoplastin protein. The phragmoplastin in such cells is visualized by examining the cells under conditions which cause the marker to become visible such as by fluorescent microscopy. 
     The present invention also relates to isolated DNA molecules which encode phragmoplastin, and cells transformed with DNA which encodes phragmoplastin, particularly DNA which encodes chimeric phragmoplastin proteins.

BACKGROUND OF THE INVENTION

The cell plate is a disc-like structure that is present only in dividingplant cells. Since cell plates are found only in plant cells, it ishoped that herbicides which directly and specifically block or interferewith cell plate development will be specific to plants and lesshazardous to animals that may be exposed to such herbicides.Unfortunately, identifying such herbicides is hampered by a scarcity oftools and methods for rapidly and easily monitoring the effect of suchherbicides on cell plate development.

Currently cell plate development is examined using phase contrastmicroscopy. However, some of the early stages of cell plate formationare difficult to examine using this tool. Cell plate development is alsomonitored by microscopic techniques in which the cells are first fixedand then incubated with aniline blue to stain the callosepolysaccharide, which is a component of the cell wall. However, becausecallose is not deposited on the cell plate until the later stages ofcell plate development, this technique also is not useful to monitor theearly stages of cell plate formation and development.

Thus, it would be desirable to have additional tools and methods forexamining the early stages of cell plate formation particularly indetermining the effect of herbicides on cell plate development. Toolsthat do not require fixation or staining or other lengthy steps wouldalso be desirable.

SUMMARY OF THE INVENTION

The present invention provides new tools and methods for identifyingherbicides and potential herbicides which affect cell plate formationand development. A new protein, phragmoplastin, has been discoveredwhich is associated with cell plate membrane vesicles duringcytokinesis. By visualizing the phragmoplastin, one is able to examinethe development of the cell plate particularly in response to herbicidesor potential herbicides. One method of visualizing phragmoplastinemploys immunocytochemical techniques with anti-phragmoplastinantibodies. Another method of visualizing phragmoplastin employs cellswhich are transformed with a DNA molecule which encodes a chimericphragmoplastin protein comprised of phragmoplastin and a luminescent tagor protein, fused to the phragmoplastin protein. The phragmoplastin insuch cells is visualized by examining the cells under conditions whichcause the marker to become visible such as by fluorescent microscopy.

The present invention also relates to isolated DNA molecules whichencode phragmoplastin, and cells transformed with DNA which encodesphragmoplastin, particularly DNA which encodes chimeric phragmoplastinproteins.

DESCRIPTION OF THE FIGURES

FIG. 1 is a nucleotide sequence, Seq. ID No. 3, of thephragmoplastin-encoding cDNA of plasmid pPDL5;

FIG. 2 is a nucleotide sequence, Seq. ID. No. 5, of thephragmoplastin-encoding cDNA of plasmid pPDL12;

FIG. 3 is the predicted amino acid sequence, Seq. ID No. 4, of thephragmoplastin encoded by the cDNA of plasmid pPDL5 as shown in FIG. 1;

FIG. 4 is the predicted amino acid sequence, Seq. ID No. 6, of thephragmoplastin encoded by the cDNA of plasmid pPDL12 as shown in FIG. 2;

FIG. 5 is a map of plasmid pBI121 -EGP.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a new protein, phragmoplastin,which is spatially and temporally associated with the cell platemembrane during all stages of cell plate development has beendiscovered. By visualizing the phragmoplastin, one is able to examinethe development of the cell plate particularly in response to herbicidesor potential herbicides. The present invention relates to differentmethods of examining the effect of potential herbicides on cell platedevelopment. In one embodiment, growing plant cells are treated with theherbicide. Then immunocytochemical methods which employanti-phragmoplastin antibodies are used to visualize the distribution ofthe phragmoplastin within the cell and to determine whether theherbicide inhibits formation or development of the cell plate. In asecond embodiment, the herbicide is administered to living transgenicplant cells comprising a DNA molecule which encodes the chimericphragmoplastin protein. The effect of the herbicide on cell plateformation and development is determined by microscopy to visualize thedistribution of the chimeric protein in transgenic cells that have beentreated with the herbicide.

Phragmoplastin

On the basis of the predicted amino acid sequences depicted in FIGS. 3and 4 and SEQ.ID NOS. 4 and 6, respectively, phragmoplastin has acalculated molecular weight of about 68.3 kDa. The phragmoplastinproteins depicted in SEQ.ID. NOS. 4 and 6 have three consensus sequenceswhich are known to be involved in GTP binding: the G1 region G(X)₄GK(ST) at position 41-48, the G3 region DX₂ G at position 142-145; andthe G4 region (TN)(KQ)XD at position 211-214. All three GTP bindingsequences are in the N-terminus region of phragmoplastin. The N-terminusof the phragmoplastin also comprises a self-assembly motif, whichincludes amino acid residues 49-93 of the phragmoplastin proteinsdepicted in SEQ. ID. NOS. 4 and 6. The phragmoplastin proteins also havea putative calcium/calmodulin-dependent protein kinase andcAMP-dependent protein kinase A site in the C-terminus, which is locatedat position 574 of the phragmoplastin proteins depicted in SEQ.ID.NOS. 4and 6. Phragmoplastin has a 41.6% homology with the animal proteindynamin. Phragmoplastin, however, lacks the C-terminal proline-richdomain of dynamin. Phragmoplastin also lacks a transmembrane bindingdomain as found in dynamin.

The term "phragmoplastin" as used herein includes proteins whichcomprise the amino acid sequence of FIG. 3 and SEQ. ID. NO 4 and theamino acid sequence of FIG. 4 and SEQ. ID. NO.6. Phragmoplastin alsoincludes proteins which have conservative amino acid substitutions inthe sequences of SEQ.ID.NOS 4 and 6, proteins which are allelic variantsof proteins comprising the amino acid sequences of SEQ. ID. NOS. 4 and6, and proteins comprising amino acid sequences that are 80% homologous,preferably 90% homologous, more preferably 95% homologous to thesequences of SEQ. ID. NO 4 or SEQ.ID. NO. 6.

It has been discovered that phragmoplastin is localized in theperinuclear region of meristemic plant cells and is not concentrated inany specific areas during the early stages of the cell cycle. Duringearly anaphase, when cell plate formation begins, phragmoplastinredistributes to the equator of the cell, where it is present across theentire cell plate disc within the phragmoplast cylinder. As the cellplate grows out toward the parental cell wall, phragmoplastinredistributes to the growing edge of the cell plate. On the basis of thestructure of phragmoplastin and the redistribution of phragmoplastin tovarious areas of the cell during cytokinesis, it is believed thatphragmoplastin plays a role in cell plate development and cell wallformation.

Because of the spatial and temporal association between phragmoplastinand the cell plate, phragmoplastin is a useful marker for cell plateformation and development in growing plant cells. Accordingly,antibodies to phragmoplastin are useful reagents for monitoring theeffect of herbicides on cell wall formation and cell wall development.In addition, transformed cells that comprise a DNA molecule whichencodes a chimeric protein comprising the phragmoplastin protein fusedto a luminescent peptide or protein tag are also useful for monitoringthe effect of herbicides on cell plate development in living cells. Suchcell lines are particularly useful for screening for herbicides thatbind to phragmoplastin or that specifically block redistribution ofphragmoplastin in the cell.

Preparation of Phragmoplastin and Phragmoplastin Antibodies

Preferably, phragmoplastin is prepared using standard recombinant DNAmethodology. Such methodology typically involves introducing apolynucleotide, preferably a DNA molecule, which encodes thephragmoplastin protein into a host cell. Following introduction of thepolynucleotide into the host cell, the phragmoplastin encoding sequencesis expressed in the host cell such that phragmoplastin protein moleculesare formed in the cell.

Preferably, the introduced polynucleotide also comprises a promoter,more preferably an inducible promoter, operably linked to thephragmoplastin encoding sequence. Preferably, the polynucleotide furthercomprises a sequence which encodes a tag to facilitate isolation of thephragmoplastin protein such as, for example, an affinity tag and/or anepitope tag. Preferably, the tag sequences are at the 5' or 3' end ofthe phragmoplastin-encoding sequence. The polynucleotide preferably alsocomprises nucleotide sequences that encode a replication origin and aselectable marker.

The polynucleotide comprising the phragmoplastin-encoding sequence isintroduced into the host cell by conventional methods, such as, bycloning the polynucleotide into a vector and by introducing the vectorinto the host cell by conventional methods. Suitable host cells include, for example, bacterial cells, yeast cells, mammalian cells, and plantcells. The DNA sequences of the introduced polynucleotide are thenexpressed in the host cell. Thereafter the expressed protein is isolatedfrom the host cell using conventional methods such as for example,chromatography and gel electrophoresis.

Although less preferred, phragmoplastin can also be prepared usingconventional synthetic biochemical techniques, such as solid phasepeptide synthesis.

In a preferred method, phragmoplastin and anti-phragmoplastin antibodieswere prepared using the following procedures.

Cloning cDNA Molecules which Encode Phragmoplastin Protein from DividingPlant Cells

cDNA clones which encode phragmoplastin were obtained using thefollowing primers:

5'-CCI(AC)G(AC)GG(AT)(AT)(GC)TGGIAT(TC)G-TIAC-3', SEQ.ID.NO. 1 and5'-TTT(TG)GTIA(AT)(AG)ACICC(ATG)ATIGT-3', SEQ.ID. NO.2.

and soybean nodule poly(A) RNA, that had been isolated from 15-day-oldroot nodules using oligo (dT). The isolated poly(A)RNA was then reversetranscribed into cDNA using oligo(dT). PCR amplification of theresulting product was performed as 45 cycles at 94° C. for 40 seconds;55° C. for 40 seconds; and 72° C. for 1 minutes The resulting 500 basepair fragment obtained was blunt-end ligated into the SmaI site of pUC19and used as a probe to screen a λ-ZapII soybean nodule cDNA library.Four positive clones were identified after screening 1×10⁶ plaques. Thepositive clones were excised and ligated into plasmids obtained fromStratagene and sequenced using the procedure of Sanger et al, 1977, inProc. Nat. Acad Sci. USA 74:5463-5467. Sequence analysis indicated thatthree of these clones, including the longest clone pPDL5, are encoded bythe same gene. The other clone, pPDL12, is encoded by a different gene.The nucleotide sequence of the pPDL5 clone is shown in FIG. 1 and SEQ.ID. NO. 3. The nucleotide sequence of the pPDL12 clone is shown in FIG.2 and SEQ. ID. NO. 5.

As shown in FIGS. 1 and 2, both pPDL5 and pPDL12 contain nearlyfull-length cDNA inserts with poly(A) tails and with 5' non-codingregions of 126 base pairs (pPDL5) and 159 base pair (pPDL 12). Each ofthese two nucleotide sequences contains a long open reading frame of1930 base pairs corresponding to a protein of 610 amino acids in lengthwith a calculated molecular weight of 68.3 kDa. The two clones differ inthe N-terminal and C-terminal non-coding regions, but share 98% homologyin their coding-regions.

Polynucleotide molecules which encode phragmoplastin protein include,for example, the nucleotide sequences of SEQ. ID. NO. 3 or SEQ.ID.NO.5or nucleotide sequences which hybridize under stringent conditions tothe sequences of SEQ.ID. NOS. 3 or 5. As used herein, the term"stringent conditions" means hybridization will occur if there is atleast 90% identity between the polynucleotide sequence and one ofSEQ.ID. NO. 3 or 5. Thus, the polynucleotide molecules which encodephragmoplastin include, for example, DNA, which DNA includes cDNA,genomic DNA, and synthetic DNA, and RNA sequences encoding allelicvariant forms of the peptides encoded by the cDNA of SEQ. ID. NOS. 3 and5. Preferably the DNA or RNA is provided in a purified or isolated form.The polynucleotide molecules which encode phragmoplastin optionallyencode a marker sequence which allows for purification of thephragmoplastin, such as for example an affinity tag or epitope tag. Thepolynucleotide molecules which encode phragmoplastin optionally encode asequence which encodes a luminescent peptide or protein fused to thecoding sequence for phragmoplastin.

Purification of Phragmoplastin from E.coli Transformed with a cDNAMolecule that Encodes Phragmoplastin

The Bg/II-KpmI fragment of pPDL12 was cloned in frame into theBamHI-KpnI site of bacterial expression vector pRSETC, which had beenobtained from Invitrogen.

The resulting construct pEPDL-12 contains anisopropyl-B-D-thiogalactopyranoside (IPTG)-regulatable promoter operablylinked to a coding sequence for a polyhistidine metal binding domainfused to the coding sequence of the phragmoplastin protein. Theconstruct was then transformed into E.coli Lys-s cells. The cells weregrown in Luria-Bertani (LB) medium to identify transformants. Expressionof the phragmoplastin protein was induced by adding 0.5 mM IPTG to themedium of the transformed cells. Phragmoplastin protein was purifiedfrom a solubilized cell pellet by chromatography on a Nickel(Ni) chelatecolumn obtained from Invitrogen. The Ni- affinity chromatography wasconducted under non-denaturing conditions according to themanufacturer's instructions. The protein eluted from the Ni column wasfurther purified by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The gel-purified phragmoplastin protein wascut out from the polyacrylamide gel and ground in liquid nitrogen toprovide the gel-purified phragmoplastin.

A single band of protein with an apparent molecular weight of 72 kDa wasobtained after Ni-affinity chromatography. The molecular weight of theexpressed protein results from fusion of a 35 amino acid polyhistidinemetal binding domain and a four amino acid linker sequence to theN-terminus of the expressed phragmoplastin protein molecules. Theexpressed protein also has the ability to hydrolyze GTP without therequirement for any other protein factors. Thus, phragmoplastin hasintrinsic GTPase activity.

Preparation of Antibody to Phragmoplastin

To prepare antibodies, the gel-purified phragmoplastin was directlyinjected into rabbits using conventional techniques. The animals werebled and the serum subjected to protein A gel chromatography asdescribed in Harlow and Lan, "Antibodies," A Laboratory Manual. ColdSpring Harbor Laboratory, 1988 to provide anti-phragmoplastinantibodies.

Assaying for Phragmoplastin at Different Stages of the Cell Cycle

The anti-soybean antiphragmoplastin antibodies prepared as describedabove were used to assay for phragmoplastin in tobacco BY-2 cellssynchronized at S phase, metaphase, and cytokinesis by the methods ofAsada and Shibaoka as described in J. Cell Sci., 107: 2249-2257, 1994,and in cells obtained from soybean root tips. A band with a molecularmass of about 68 kDa was detected with the anti-soybean phragmoplastinantibody in the lanes containing proteins extracted from both dividingsoybean root cells and dividing tobacco BY-2 cells, indicating thatphragmoplastin is conserved in plants. Thus, anti-soybean phragmoplastinantibodies are able to bind to phragmoplastin from various species ofplants. Phragmoplastin in tobacco cells and soybean was most abundant inthe microsomal membrane fractions with a very low concentration in thecytosolic fractions. The ratio of phragmoplastin in microsomes and inthe cytosol did not change during the cell cycle, indicating thatphragmoplastin is associated primarily with membranes at all stages ofthe cell cycle.

Methods for Examining Cell Plate Development

Because phragmoplastin is spatially and temporally associated with thecell plates in dividing cells, polyclonal and monoclonalanti-phragmoplastin antibodies enable one to monitor the distribution ofphragmoplastin in dividing plant cells and to examine cell platedevelopment. Preferably, the antibodies are used in an immunolabelingmethod. The antibodies are used to immunolabel dividing cells, such asfor examples cells that have been obtained from root tips or plant cellcultures. During the immunolabeling procedure, the cells are fixed,permeablized and incubated with the anti-phragmoplastin antibody.Optionally, the cell walls are removed prior to incubation with theanti-phragmoplastin antibody to improve labeling. Then, the cells areincubated with a second antibody which binds to the anti-phragmoplastinantibody and which carries a fluorescent or chemiluminescent tag. Thecells are then visualized under a microscope and the distribution of theimmunolabeled phragmoplastin in the cell determined.

The following examples of methods which use phragmoplastin antibodies toexamine cell plate formation and development in dividing cells areillustrative and are not meant to limit the scope of the invention.

EXAMPLE 1

Immunolabeling Cell Plates in Dividing Cells

Four-day-old soybean root tips or eleven-day-old nodules were fixed in4% paraformaldehyde in buffer containing 60 mM PIPES, 25 mM HEPES, 10 mMEGTA, 2 mM MgCl₂, pH 7.0 (PHEM buffer) for 2 hours at room temperatureand then washed with washing buffer containing 10 mM MES, pH 5.7, 30 mMCaCl₂, 0.1% BSA, 5 mM 2-mercaptoethanol, 0.4 M mannitol for 30 minutes.The plant tissues were then digested in a solution containing 2%cellulase from Calbiochem, 1% macerozyme R-10 from Ykult, Honsha, Japan,0.5% pectolyase Y23 from Karlan in washing buffer for 15 minutes. Afterrinsing in PHEM buffer, the plant tissues were squashed betweencoverslips to release the individual cells. The cells were air dried for10 minutes and then permeablized with 0.5% Triton X-100 in PHEM for 5minutes. The phragmoplastin in the cells was then immunolabeled usingaffinity-purified anti-phragmoplastin IgG as the first antibody andfluorescein isothiocyanate (FITC)-conjugated anti-rabbit goat IgG as asecond antibody. Pre-immune serum was used as the first antibody in thecontrol group. DNA was stained with 4'6'-diamidino-2-phenylindole (DAPI)at a concentration of 10 μg/ml for 5 minutes at room temperature. Cellswere visualized in a confocal or fluorescent microscope equipped withexcitation/emission filter sets for FITC. Photographs of the intactcells were taken in phase contrast, and using appropriate filters forFITC and DAPI stainings.

In metaphase when the two sets of chromosomes just begin to separate, nocell plate could be visualized by anti-phragmoplastin antibody labelingor phase contrast microscopy. However, by late anaphase or earlytelophase, the inter-zone region of the dividing plant cell could bevisualized with anti-phragmoplastin antibodies. In comparison, phasecontrast microscopy did not enable visualization of the cell plate inanaphase. When the cells reached telophase, the cell plate could bedistinguished in the phase contrast image. During telophase theanti-phragmoplastin antibody first reacted with the entire width of thecell plate and then with the periphery of the cell plate. When the twodaughter cells separated completely, the anti-phragmoplastin antibodygave a perinuclear particulate staining pattern. No phragmoplastinfluorescence was observed on the newly formed plasma membranes or cellwalls of these two daughter cells.

The anti-phragmoplastin antibodies enabled the cell plate to bevisualized from a point at the earliest stages of cell plate formationin early anaphase until the very latest stages of cell plate formationwhen the cell plate reaches the parental cell wall.

EXAMPLE 2

Immunolabeling the Cell Plate and Microtubules in Dividing Cells

Intact cells from soybean root tips were treated as described above inexample 1 except that the permeabilized cells were also incubated withmouse anti-tubulin IgG, obtained from Amersham and withtetramethylrhodamine isothiocyantate (TRITC)-conjugated goat anti-mouseIgG from Sigma to stain the microtubules. The double-labeled cells werevisualized in a confocal microscope equipped with dualexcitation/emission filter sets for both FITC and TRITC. Photographs ofthe cells were taken in phase contrast, and using white light and bluelight and appropriate filters for FITC, TRITC and DAPI fitted to a Zeissmicroscope.

A metaphase spindle and phragmoplasts at different stages of cytokineseswere clearly distinguished with microtubule labeling. The bright line ofphragmoplastin fluorescence first appeared in cells at anaphase. As thecell plate grew outwards, both the phragmoplastin protein fluorescenceand the microtubule fluorescence decreased in the middle of thephragmoplasts. However, a bright fluorescence line of phragmoplastinprotein was still visible at the end of cytokinesis when microtubulefluroescence had almost disappeared. These results indicate that thedistribution of phragmoplastin is related to but independent frommicrotuble formation during cytokinesis. Thus, anti-phragmoplastinantibodies allow one to visualize and characterize events in cell plateformation and development that cannot be examined using anti-tubulinantibodies.

EXAMPLE 3

Immunolabeling the Cell Plate and Microtubules in Dividing Cells

Three-day-old tobacco BY-2 cells were fixed in 4% paraformaldehyde in 60mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgC12, pH 7.0 (PHEM) for Ihours at room temperature and then washed on one layer of Miracloth withbuffer containing 10 mM MES, pH 5.7, 30 mM CaCl₂, 0.1% BSA, 5 mMβ-mercaptoethanol and 0.4 M mannitol. Cells were then digested for 15minutes in a solution containing 1% cellulase from Calbiochem, SanDiego, Cailf. and 0.1 % pectolyase Y23 in washing buffer. After rinsingwith PHEM buffer, cells were transferred to poly-L-lysine-coated slidesand air dried for 10 minutes. Immunofluorescence microscopy was thencarried out using the affinity-purified anti-soybean phragmoplastin IgGas the first antibody and the fluorescein isothiocyanate(FITC)-conjugated anti-rabbit goat IgG as a second antibody. Pre-immuneserum was used as the first antibody in the control group. Fordouble-labeling experiments, mouse anti-tubulin IgG from Amersham andtetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouseIgG from Sigma were used to stain microtubules. DNA was stained withDAPI at a concentration of 10 μg/ml for 5 minutes at room temperatureand microscopy conducted as described above in example 2.

Immunofluorescence microscopy using anti-soybean phragmoplastinantibodies demonstrated that phragmoplastin is associated with cellplates in tobacco BY-2 cells. In the early stages of cell plateformation, phragmoplastin was present across the entire cell plate discwithin the phragmoplast proper. As the cell plate grew outward,phragmoplastin was concentrated more on the growing margins of the cellplate whereas the concentration in the middle of the cell platedecreased. A ring-like, annular distribution pattern of phragmoplastinfluorescence was observed at this stage in many cells. As cell plateformation was completed, phragmoplastin disappeared, and only aperinuclear diffused pattern of phragmoplastin was visible in thenondividing cells. Thus, antibodies to phragmoplastin enable the cellplates to be marked and to differentiate the early and late stages ofcell plate formation and development.

Preparation of a Transgenic Cell Line

Transgenic cells which express a chimeric phragmoplastin protein thatcan be visualized in living cells by microscopy are prepared bytransforming plant cells with a vector, such as for example the plantvector Agrobacterium which contains a DNA molecule that encodesphragmoplastin linked at its N-terminus or C-terminus to a luminescentpolypeptide. In a preferred embodiment, cultured plant cells with avector containing a DNA molecule having a coding sequence forphragmoplastin protein linked to a coding sequence for the greenfluorescence protein (GFP) as described below to provide transgenic celllines that express the chimeric phragmoplastin protein,GFP-phragmoplastin.

The plasmid pBI-35S-mGFP, which contains a nucleotide sequence encodingthe GFP was obtained from J. Haesoloff, MRC Laboratory, Cambridge, UK. Aplasmid containing the GFP encoding sequence can also be obtained fromClonetech Labs, Palo Alto, Calif. The plasmid was amplified usingstandard techniques and then the GFP coding region was cloned intoplasmid pUC-GFP44 obtained from Dr. Z. Yang, Ohio State University,Columbus, Ohio. The GFP coding region was then excised and ligated intothe XbaI-SacI sites of PBI221-JR, also obtained from Dr. Z. Yang, toprovide plasmid pE-GFP44, which contained a Cauliflower mosaic virus 35Spromoter, a GFP coding sequence with a Bg1II site at the C-terminal end,and a nopaline synthase terminator. The Bg1II-SacI fragment ofphragmoplastin coding sequence as shown in SEQ. ID. NO. 5 was removedfrom plasmid pEPDL12, prepared as described above, and cloned into theBg1II-SacI sites of pE-GFP44 to provide the plasmid pE-GFP44-PDL-BS. TheXbaI-SacI fragment of the plasmid pE-GFP44-PDL-BS was finally cloned into XbaI-SacI sites of plasmid pBI121-MLB-PDL12, obtained from ClonetechLabs, Palo Alto, Calif. to provide plasmid pBI121-EGP, which is depictedin FIG. 5. Plasmid pBI121-EGP contained a 35S promoter, a codingsequence for a GFP-phragmoplastin, and a nopaline synthase terminator.Plasmid pBI 121 -EGP was transformed into Agrobacterium tumefaciensLBA4404 using a freeze-thaw method.

Transformation of BY-2 cells was performed using the procedure of An etal., "Binary Vectors", In Plant Molecular Biology Manual, pp A3/1, 1992.A five ml liquid culture of 3-day-old BY-2 cells was mixed with 100 μLof Agrobacterium tumefaciens LBA4404 harboring the plasmid pBI121-EGP.The cells were allowed to grow in Petri plates at 25° C. without shakingfor 2 days. Cells were then filtered on one layer of Miracloth andwashed to remove most of the bacteria. The washed cells were then platedon solid Murashige and Skoog medium containing 300 μg kanamycin and 500μg of carbenicillin. Transgenic calli usually emerged after 2-3 weeks.The calli were then transferred to new antibiotic-containing plates.

Transgenic cells (20 mg fresh weight) from different calli were digestedfor 20 minutes at room temperature in 200 μL of enzyme solution of 1%cellulase in 10 mM Mes, pH 5.7 and 0.38 M sorbitol. Cells were thencentrifuged for 2 minutes in a microcentrifuge at room temperature. Theenzyme solution was removed and the cells were washed with 200 μL ofwashing solution containing 1% Triton X-100 in PHEM. After pelleting,the cells were directly solubilized in SDS sample buffer and processedfor SDS-PAGE and protein gel blotting. All transgenic lines except onewere found to express GFP-phragmoplastin, which has a molecular weightof approximately 90 kDa as determined by SDS-gel electrophoresis. About50% of the kanamycin-resistant cell lines tested were found to expressGFP-phragmoplastin at a level 2 to 5 fold higher than nativephragmoplastin protein and three lines overexpressed theGFP-phragmoplastin at levels that were 5 to 10 fold higher than thelevels of native phragmoplastin in untransformed BY-2 cells.

GFP-phragmoplastin is stable in the transgenic BY-2 cells. Even though adegradation product of the native phragmoplastin was found in most ofthe preparations, no degradation products of GFP-phragmoplastin weredetected by protein gel blots analysis. The degradation products of thenative phragmoplastin were not detected if cell extract was made in thepresence of proteinase inhibitors. Similar to the native phragmoplastin,the GFP-phragmoplastin was predominantly associated with the microsomalmembrane fraction.

Fluorescence microscopy showed that the transgenic cells overexpressingGFP-phragmoplastin at 2 to 10 fold higher than native phragmoplastinexhibited bright green fluorescence on the cell plate duringcytokinesis, whereas no green fluorescence was observed in untransformedBY-2 cells. Control BY-2 cells, which were transformed with GFP aloneexhibited a green fluorescence throughout the cytoplasm but did notexhibit a defined fluorescent line in the cell plate region. Thus, thetargeting of the GFP-phragmoplastin to the cell plate is the function ofthe phragmoplastin part of the chimeric protein.

The development of the cell plate was examined in transgenic cells whichwere prepared as described above and which overexpressGFP-phragmoplastin to levels 5 to 10× greater than the levels of nativephragmoplastin in untransformed BY-2 cells. All transgenic cells weregrown on solid medium or in liquid medium. Then 100 μL of liquid culturecells were mixed with 100 μL of medium in a culture well on a slide. Thedevelopment of the cell plate in the cells was examined over time in anindividual cell by visualizing the GFP-phragmoplastin fluorescence underan inverted epifluoresence microscope equipped with an FITC filter setand taking photographs of the cell at different stages in the cellcycle. To reduce photobleaching, an excitation light was turned on onlywhen photographs were taken.

In the early stages of cell plate formation, the GFP-phragmoplastinappeared as a dense short fluorescent line in the center of the dividingcells. The GFP-phragmoplastin fluorescence appeared soon after the onsetof anaphase and gradually increased in intensity forming a short linewithin 30 minutes. As the cell plate expanded outward, the fluorescencein the center of the cell plate decreased while the intensity of thefluorescence at the margin of the cell plate increased. Thereafter, aring-like, annular fluorescence was observed in the cell. As the cellplate enlarged, there was a gradual decrease of the overall intensity ofthe GFP-phragmoplastin fluorescent ring. After the cell plate reachedthe parental cell wall, the green fluorescence gradually disappeared.The fluorescence of GFP-phragmoplastin on the cell plate remained stableup to 2 hours. Thus, the chimeric protein GFP-phragmoplastin follows thesame pattern of distribution during cytokinesis as nativephragmoplastin.

During the initial stages of cell plate formation, theGFP-phragmoplastin fluorescence was confined to the center of thephragmoplast proper. During the later stage, when the cell plateextended beyond the phragmoplast proper, the GFP fluoroescenceredistributed to the growing edge of the cell plate. Thus,GFP-phragmoplastin, like native phragmoplastin is associated with thecell plate.

In the cells which overexpressed GFP-phragmoplastin to levels that weremore than 5 to 10 fold greater than native phragmoplastin, the time forthe cell plate to fuse with the prenatal cell wall was greater than innon-transformed cells and often resulted in the formation of an obliquecell plate. The fact that these transgenic cells continued to elongate,irrespective of the orientation of cell division, indicates that inthese particular cell lines the plane of cell division is uncoupled fromthe direction of cell elongation.

Transgenic cells which overexpress a phragmoplastin protein fused to aluminescent marker permit observation of cell plate development in aliving cell continuously and throughout the entire process ofcytokinesis. Accordingly, such transgenic cell lines provide a researchtool which avoids many of the problems that are encountered whenconventional research tools are used to examine cell plate formation.For example, use of the transgenic cell lines eliminates the need to fixthe cells, which can occasionally distort subcellular structures. Use ofthe transgenic cell line also avoids the lengthy staining steps whichare often required to examine cell plate development. Use of thetransgenic cell line also permits examination of cell plate developmentin a single cell, and as such, avoids any problems that may result fromobserving a dynamic process in a multitude of cells which appear to bein different stages of the cell cycle. Thus, it is less likely thatshort-lived steps in cell plate development will be overlooked when suchtransgenic cell lines are used to examine cell plate development.

Examining the Effect of Herbicides on Cell Plate Development

In one embodiment, the effect of herbicides whether known herbicides orherbicides on cell plate development is examined usinganti-phragmoplastin antibodies. Preferably, the antibodies are used inan immunolabeling method which commences with application of theherbicide to dividing plant cells. Preferably various concentrations ofthe herbicide are applied to determine the optimum concentration forinhibiting cell plate development. Although the herbicide may be appliedto a whole plant or plant segment that comprises dividing cells, it ispreferred that the herbicidal compound be added directly to the mediumof plant cell cultures. The use of plant cell cultures eliminates thestep of dissociating the cells of the growing plant. In addition, theuse of plant cell cultures allows synchronization of the dividing cellsto the same stages of mitosis. When applied to synchronized cells, it ispreferred that the herbicidal compound be applied before anaphase.Thereafter, the cells typically are fixed, permeablized and incubatedwith the anti-phragmoplastin antibody. Optionally, the cell walls areremoved prior to incubation with the anti-phragmoplastin antibody toenhance labeling with the antibody. Preferably, the cells are thenincubated with a second antibody which binds to the anti-phragmoplastinantibody and which carries a fluorescent or chemiluminescent tag. Thecells are then visualized under a microscope and the distribution of theimmunolabeled phragmoplastin in the cell is determined to identifyherbicides which inhibit cell plate formation and development and tocharacterize the stages in cell plate development where such herbicidesexert their effect.

In a second embodiment , the herbicide is applied to transgenic plantcells that express a chimeric phragmoplastin protein that comprisesphragmoplastin fused to a luminescent peptide or protein tag.Thereafter, the distribution of the chimeric protein within the livingcell is continuously monitored to determine the effect of the herbicideon cell plate development. To permit easy visualization of the chimericprotein, it is preferred that the transgenic cells overexpress thechimeric protein to levels that are 5 to 10 fold higher than the levelof native phragmoplastin. Preferably, the transgenic cells aresynchronized prior to treatment with the herbicide. Preferably, theherbicide is applied to the cells at various stages in the cell cycle,including metaphase, anaphase, and telophase. Preferably, thedistribution of the chimeric protein is monitored over time in livingcells that have not been fixed or permeabilized. Preferably, thedistribution of the chimeric protein is monitored using microscopy, suchas for example, epifluorescence microscopy as described in examples 3and 4 or wide-field microscopy followed by image deconvolution asdescribed by Gens J. S. et al. in Protoplasma 194:215-230, 1996 which isincorporated herein by reference

EXAMPLE 4

Effect of Caffeine on Cell Plate Development

Caffeine inhibits cell plate development in plant cells. To determinewhether caffeine affects the early stages or later stages of cell platedevelopment in living cells, 100 μL of liquid culture transgenic BY-2cells that were overexpressing GFP-phragmoplastin, prepared as describedabove, were mixed with 100 μL of medium in a culture well. One of thedividing transgenic cells was then located under an invertedepifluorescence microscope and the presence of the GFP-phragmoplastinfluorescence at the center of the cell and on the cell plate wasrecorded. Then 50 μL of caffeine was added to the medium in the culturechamber to a final concentration 10 mM. Thereafter the distribution ofthe GFP-phragmoplastin fluorescence in the cell was monitored andrecorded by photographing the same cell at different time intervalsfollowing addition of the caffeine to the medium. Photographs were takenfor 2 hours following addition of the caffeine.

EXAMPLE 5

Effect of Taxol on Cell Plate Development

The effect of taxol on cell plate development was determined asdescribed in example 4 for caffeine, except that 10 μL of taxol ratherthan caffeine was added to the medium to a final concentration 50 mM.

As shown by the photographs of the transgenic cells of example 4,caffeine treatment had no apparent effect on the distribution ofphragmoplastin on the cell plate during the early stages of cell platedevelopment. However, at the later stages of cell plate development,caffeine treatment blocked the increase in phragmoplastin at the growingmargins of the cell plate. Thereafter, the phragmoplastin fluorescenceon the cell plate started to disappear from the periphery and accumulatein the center of the cell plate. This is in sharp contrast to thecontrol transgenic cells in which the phragmoplastin fluorescence in theperiphery increases while the phragmoplastin fluorescence in the centerdecreases as the cell plate extends to meet the prenatal cell wall.

As shown by the photographs of the transgenic cells of example 5, there-distribution of phragmoplastin on the cell plate in the taxol-treatedtransgenic cells was very different from the re-distribution ofphragmoplastin on the cell plate in control transgenic cells andcaffeine-treated transgenic cells. Taxol immediately stopped the outwardredistribution of phragmoplastin. The green ring of GFP-phragmoplastinfluorescence was present in the cells of example 5 throughout the 2 hourobservation period. There was no change in shape or size of this ring.It is believed that that the effect of the taxol on cell platedevelopment and phragmoplastin distribution in the

Numerous modifications and variations of the present invention arepossible in light of the above teachings and are, therefore, within thescope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 15                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 21                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CCNMGMGGWW STGGNATYGT NAC           - #                  - #                    23                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 7                                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 13                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 19                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TTTKGTNAWR ACNCCDATNG T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2134 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 127..1956                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GCACCAAGCA CCAACAACGC TTTAGCTCTC TCTCTTCTCT CCATAACCGC CA -             #CCGGCGAT     60                                                                 - - CTGGCACTAA CAGATCGCCG CTGCTACATC TGAACCCGAT CCAGCCAACA GA -            #TCTCTCCA    120                                                                 - - ATTCAA ATG GAG AAT CTA ATC TCT TTG GTC AAC - #AAA ATC CAG AGA GCT           168                                                                              Met Glu Asn Leu Ile Ser Le - #u Val Asn Lys Ile Gln Arg Ala                     1         - #      5            - #      10                           - - TGC ACC GCC TTA GGT GAC CAC GGC GAA AAC AG - #T GCA CTC CCC ACA CTA          216                                                                       Cys Thr Ala Leu Gly Asp His Gly Glu Asn Se - #r Ala Leu Pro Thr Leu            15                 - # 20                 - # 25                 - # 30       - - TGG GAC TCT CTC CCC GCC ATC GCC GTC GTC GG - #A GGC CAG AGC TCA GGA          264                                                                       Trp Asp Ser Leu Pro Ala Ile Ala Val Val Gl - #y Gly Gln Ser Ser Gly                            35 - #                 40 - #                 45              - - AAG TCC TCC GTC TTG GAG AGC GTT GTC GGC AA - #A GAT TTC TTA CCT CGT          312                                                                       Lys Ser Ser Val Leu Glu Ser Val Val Gly Ly - #s Asp Phe Leu Pro Arg                        50     - #             55     - #             60                  - - GGA TCA GGT ATT GTT ACG CGA CGA CCG CTC GT - #G TTG CAG CTT CAC AAG          360                                                                       Gly Ser Gly Ile Val Thr Arg Arg Pro Leu Va - #l Leu Gln Leu His Lys                    65         - #         70         - #         75                      - - ATT GAA GAG GGA AGC AGA GAG TAC GCG GAG TT - #C CTC CAC CTC CCG AGG          408                                                                       Ile Glu Glu Gly Ser Arg Glu Tyr Ala Glu Ph - #e Leu His Leu Pro Arg                80             - #     85             - #     90                          - - AAG AGG TTC ACC GAT TTT GTT GCT GTG AGG AA - #G GAG ATT CAA GAC GAA          456                                                                       Lys Arg Phe Thr Asp Phe Val Ala Val Arg Ly - #s Glu Ile Gln Asp Glu            95                 - #100                 - #105                 - #110       - - ACT GAT AGA GAG ACT GGA CGA ACC AAA CAA AT - #T TCT ACT GTT CCC ATT          504                                                                       Thr Asp Arg Glu Thr Gly Arg Thr Lys Gln Il - #e Ser Thr Val Pro Ile                           115  - #               120  - #               125              - - CAT CTT AGT ATA TAC TCT CCC AAT GTT GTT AA - #C TTG ACA CTC GTT GAT          552                                                                       His Leu Ser Ile Tyr Ser Pro Asn Val Val As - #n Leu Thr Leu Val Asp                       130      - #           135      - #           140                  - - CTT CCT GGG CTT ACG AAA GTA GCT GTT GAG GG - #T CAA CCG GAT AGT ATT          600                                                                       Leu Pro Gly Leu Thr Lys Val Ala Val Glu Gl - #y Gln Pro Asp Ser Ile                   145          - #       150          - #       155                      - - GTG AAA GAC ATT GAG GAT ATG GTT CGC TCC TA - #C ATT GAG AAG CCG AAC          648                                                                       Val Lys Asp Ile Glu Asp Met Val Arg Ser Ty - #r Ile Glu Lys Pro Asn               160              - #   165              - #   170                          - - TGT ATA ATT TTG GCC ATT TCA CCA GCC AAT CA - #A GAT CTT GCA ACA TCT          696                                                                       Cys Ile Ile Leu Ala Ile Ser Pro Ala Asn Gl - #n Asp Leu Ala Thr Ser           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - GAT GCA ATT AAA ATT TCC CGT GAA GTG GAC CC - #T ACT GGA GAT AGG        ACC      744                                                                    Asp Ala Ile Lys Ile Ser Arg Glu Val Asp Pr - #o Thr Gly Asp Arg Thr                          195  - #               200  - #               205              - - ATT GGA GTT TTG ACA AAG ATT GAT CTT ATG GA - #C AAG GGT ACT GAT GCT          792                                                                       Ile Gly Val Leu Thr Lys Ile Asp Leu Met As - #p Lys Gly Thr Asp Ala                       210      - #           215      - #           220                  - - GTT GAT ATA TTG GAA GGA AGA GCA TAT AGG TT - #A AAG TTT CCC TGG ATT          840                                                                       Val Asp Ile Leu Glu Gly Arg Ala Tyr Arg Le - #u Lys Phe Pro Trp Ile                   225          - #       230          - #       235                      - - GGT GTT GTG AAT AGA TCA CAA CAA GAC ATA AA - #C AAG AAT GTT GAC ATG          888                                                                       Gly Val Val Asn Arg Ser Gln Gln Asp Ile As - #n Lys Asn Val Asp Met               240              - #   245              - #   250                          - - ATT GCT GCT AGG CGT AGA GAA CGT GAG TAC TT - #C AAT AGT ACC CCT GAA          936                                                                       Ile Ala Ala Arg Arg Arg Glu Arg Glu Tyr Ph - #e Asn Ser Thr Pro Glu           255                 2 - #60                 2 - #65                 2 -      #70                                                                              - - TAT AAA CAC CTT GCG AAC AGA ATG GGT TCC GA - #G CAT CTG GCG AAG        ATG      984                                                                    Tyr Lys His Leu Ala Asn Arg Met Gly Ser Gl - #u His Leu Ala Lys Met                          275  - #               280  - #               285              - - CTC TCA AAG CAT TTG GAG ACA GTA ATC AAG TC - #C AAA ATT CCT GGC ATT         1032                                                                       Leu Ser Lys His Leu Glu Thr Val Ile Lys Se - #r Lys Ile Pro Gly Ile                       290      - #           295      - #           300                  - - CAA TCT CTA ATT AAC AAA ACA ATT GCT GAA CT - #T GAA GCT GAA CTA ACT         1080                                                                       Gln Ser Leu Ile Asn Lys Thr Ile Ala Glu Le - #u Glu Ala Glu Leu Thr                   305          - #       310          - #       315                      - - CGT TTA GGA AAG CCT GTA CGA GCT GAT GCT GG - #G GGA AAG TTG TAT GCA         1128                                                                       Arg Leu Gly Lys Pro Val Arg Ala Asp Ala Gl - #y Gly Lys Leu Tyr Ala               320              - #   325              - #   330                          - - ATC ATG GAA ATA TGC CGC TCA TTT GAT CAA AT - #A TTT AAA GAC CAT CTT         1176                                                                       Ile Met Glu Ile Cys Arg Ser Phe Asp Gln Il - #e Phe Lys Asp His Leu           335                 3 - #40                 3 - #45                 3 -      #50                                                                              - - GAT GGC GTG CGG CCT GGA GGT GAT AAA ATT TA - #T AAT GTC TTT GAC        AAT     1224                                                                    Asp Gly Val Arg Pro Gly Gly Asp Lys Ile Ty - #r Asn Val Phe Asp Asn                          355  - #               360  - #               365              - - CAG CTC CCC GCT GCT TTA AAA AGG TTG CAG TT - #T GAT AAG CAG CTT TCA         1272                                                                       Gln Leu Pro Ala Ala Leu Lys Arg Leu Gln Ph - #e Asp Lys Gln Leu Ser                       370      - #           375      - #           380                  - - ATG GAA AAT ATA AGG AAA CTT ATT ACT GAA GC - #T GAT GGG TAT CAG CCT         1320                                                                       Met Glu Asn Ile Arg Lys Leu Ile Thr Glu Al - #a Asp Gly Tyr Gln Pro                   385          - #       390          - #       395                      - - CAT CTT ATA GCT CCA GAA CAA GGA TAT CGT CG - #T CTA ATT GAA TCT TCT         1368                                                                       His Leu Ile Ala Pro Glu Gln Gly Tyr Arg Ar - #g Leu Ile Glu Ser Ser               400              - #   405              - #   410                          - - CTA ATA ACT ATT AGG GGC CCT GCT GAG GCA GC - #T GTT GAT GCG GTT CAC         1416                                                                       Leu Ile Thr Ile Arg Gly Pro Ala Glu Ala Al - #a Val Asp Ala Val His           415                 4 - #20                 4 - #25                 4 -      #30                                                                              - - TCG CTG TTA AAG GAC TTG GTT CAC AAA GCT AT - #C AGT GAG ACT TTG        GAC     1464                                                                    Ser Leu Leu Lys Asp Leu Val His Lys Ala Il - #e Ser Glu Thr Leu Asp                          435  - #               440  - #               445              - - TTG AAG CAG TAT CCT GGT CTC CGG GTT GAG GT - #T GGG GCT GCT GCT GTT         1512                                                                       Leu Lys Gln Tyr Pro Gly Leu Arg Val Glu Va - #l Gly Ala Ala Ala Val                       450      - #           455      - #           460                  - - GAT TCA CTA GAA AGA ATG AGG GAT GAA AGC AA - #A AGA GCA ACA CTG CAG         1560                                                                       Asp Ser Leu Glu Arg Met Arg Asp Glu Ser Ly - #s Arg Ala Thr Leu Gln                   465          - #       470          - #       475                      - - CTA GTT GAT ATG GAG TGT GGC TAT CTG ACT GT - #T GAT TTC TTT CGG AAG         1608                                                                       Leu Val Asp Met Glu Cys Gly Tyr Leu Thr Va - #l Asp Phe Phe Arg Lys               480              - #   485              - #   490                          - - CTT CCT CAA GAT GTT GAT AAG GGT GGC AAT CC - #C ACA CAT TCA ATT TTT         1656                                                                       Leu Pro Gln Asp Val Asp Lys Gly Gly Asn Pr - #o Thr His Ser Ile Phe           495                 5 - #00                 5 - #05                 5 -      #10                                                                              - - GAT AGA TAT AAT GAT TCA TAT CTA AGG CGA AT - #T GGA ACC ACA ATT        TTG     1704                                                                    Asp Arg Tyr Asn Asp Ser Tyr Leu Arg Arg Il - #e Gly Thr Thr Ile Leu                          515  - #               520  - #               525              - - TCA TAT GTC AAT ATG GTC TGT GCT ACA CTG CG - #G AAT TCA ATT CCC AAG         1752                                                                       Ser Tyr Val Asn Met Val Cys Ala Thr Leu Ar - #g Asn Ser Ile Pro Lys                       530      - #           535      - #           540                  - - TCC ATC GTC TAT TGT CAA GTG CGG GAG GCA AA - #A CGA AGT CTA CTT GAT         1800                                                                       Ser Ile Val Tyr Cys Gln Val Arg Glu Ala Ly - #s Arg Ser Leu Leu Asp                   545          - #       550          - #       555                      - - CAC TTT TTT ACC GAG CTA GGC AAA ATG GAG AC - #C AAG CGT CTG TCA TCG         1848                                                                       His Phe Phe Thr Glu Leu Gly Lys Met Glu Th - #r Lys Arg Leu Ser Ser               560              - #   565              - #   570                          - - TTA TTG AAT GAG GAT CCT GCA ATT ATG GAA CG - #A CGT AGT GCG CTC GCA         1896                                                                       Leu Leu Asn Glu Asp Pro Ala Ile Met Glu Ar - #g Arg Ser Ala Leu Ala           575                 5 - #80                 5 - #85                 5 -      #90                                                                              - - AAG AGA CTA GAG TTA TAC CGG AGT GCA CAA GC - #T GAA ATA GAT GCA        GTT     1944                                                                    Lys Arg Leu Glu Leu Tyr Arg Ser Ala Gln Al - #a Glu Ile Asp Ala Val                          595  - #               600  - #               605              - - GCT TGG TCT AAG TAGATATATG TATGTCAGAT CACGTTTATA CG - #AGAGCCAG             1996                                                                       Ala Trp Ser Lys                                                                           610                                                                - - CAGTGTCATT ATTCATTGTT TCCCTATTTC CAGTTCATTA TTCATACTCA TT -             #TTTTGTTG   2056                                                                 - - TCATCTTATC CACTTGTATT GTCATCTTAA ATAGATGAGA CGATTCTGAA AA -            #GGGAAAAA   2116                                                                 - - AATGATTTTT TGGGTTAT             - #                  - #                      - #2134                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 610 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Glu Asn Leu Ile Ser Leu Val Asn Lys Il - #e Gln Arg Ala Cys Thr        1               5 - #                 10 - #                 15              - - Ala Leu Gly Asp His Gly Glu Asn Ser Ala Le - #u Pro Thr Leu Trp Asp                   20     - #             25     - #             30                  - - Ser Leu Pro Ala Ile Ala Val Val Gly Gly Gl - #n Ser Ser Gly Lys Ser               35         - #         40         - #         45                      - - Ser Val Leu Glu Ser Val Val Gly Lys Asp Ph - #e Leu Pro Arg Gly Ser           50             - #     55             - #     60                          - - Gly Ile Val Thr Arg Arg Pro Leu Val Leu Gl - #n Leu His Lys Ile Glu       65                 - # 70                 - # 75                 - # 80       - - Glu Gly Ser Arg Glu Tyr Ala Glu Phe Leu Hi - #s Leu Pro Arg Lys Arg                       85 - #                 90 - #                 95              - - Phe Thr Asp Phe Val Ala Val Arg Lys Glu Il - #e Gln Asp Glu Thr Asp                  100      - #           105      - #           110                  - - Arg Glu Thr Gly Arg Thr Lys Gln Ile Ser Th - #r Val Pro Ile His Leu              115          - #       120          - #       125                      - - Ser Ile Tyr Ser Pro Asn Val Val Asn Leu Th - #r Leu Val Asp Leu Pro          130              - #   135              - #   140                          - - Gly Leu Thr Lys Val Ala Val Glu Gly Gln Pr - #o Asp Ser Ile Val Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Ile Glu Asp Met Val Arg Ser Tyr Ile Gl - #u Lys Pro Asn Cys        Ile                                                                                             165  - #               170  - #               175             - - Ile Leu Ala Ile Ser Pro Ala Asn Gln Asp Le - #u Ala Thr Ser Asp Ala                  180      - #           185      - #           190                  - - Ile Lys Ile Ser Arg Glu Val Asp Pro Thr Gl - #y Asp Arg Thr Ile Gly              195          - #       200          - #       205                      - - Val Leu Thr Lys Ile Asp Leu Met Asp Lys Gl - #y Thr Asp Ala Val Asp          210              - #   215              - #   220                          - - Ile Leu Glu Gly Arg Ala Tyr Arg Leu Lys Ph - #e Pro Trp Ile Gly Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Asn Arg Ser Gln Gln Asp Ile Asn Lys As - #n Val Asp Met Ile        Ala                                                                                             245  - #               250  - #               255             - - Ala Arg Arg Arg Glu Arg Glu Tyr Phe Asn Se - #r Thr Pro Glu Tyr Lys                  260      - #           265      - #           270                  - - His Leu Ala Asn Arg Met Gly Ser Glu His Le - #u Ala Lys Met Leu Ser              275          - #       280          - #       285                      - - Lys His Leu Glu Thr Val Ile Lys Ser Lys Il - #e Pro Gly Ile Gln Ser          290              - #   295              - #   300                          - - Leu Ile Asn Lys Thr Ile Ala Glu Leu Glu Al - #a Glu Leu Thr Arg Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Lys Pro Val Arg Ala Asp Ala Gly Gly Ly - #s Leu Tyr Ala Ile        Met                                                                                             325  - #               330  - #               335             - - Glu Ile Cys Arg Ser Phe Asp Gln Ile Phe Ly - #s Asp His Leu Asp Gly                  340      - #           345      - #           350                  - - Val Arg Pro Gly Gly Asp Lys Ile Tyr Asn Va - #l Phe Asp Asn Gln Leu              355          - #       360          - #       365                      - - Pro Ala Ala Leu Lys Arg Leu Gln Phe Asp Ly - #s Gln Leu Ser Met Glu          370              - #   375              - #   380                          - - Asn Ile Arg Lys Leu Ile Thr Glu Ala Asp Gl - #y Tyr Gln Pro His Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ile Ala Pro Glu Gln Gly Tyr Arg Arg Leu Il - #e Glu Ser Ser Leu        Ile                                                                                             405  - #               410  - #               415             - - Thr Ile Arg Gly Pro Ala Glu Ala Ala Val As - #p Ala Val His Ser Leu                  420      - #           425      - #           430                  - - Leu Lys Asp Leu Val His Lys Ala Ile Ser Gl - #u Thr Leu Asp Leu Lys              435          - #       440          - #       445                      - - Gln Tyr Pro Gly Leu Arg Val Glu Val Gly Al - #a Ala Ala Val Asp Ser          450              - #   455              - #   460                          - - Leu Glu Arg Met Arg Asp Glu Ser Lys Arg Al - #a Thr Leu Gln Leu Val      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asp Met Glu Cys Gly Tyr Leu Thr Val Asp Ph - #e Phe Arg Lys Leu        Pro                                                                                             485  - #               490  - #               495             - - Gln Asp Val Asp Lys Gly Gly Asn Pro Thr Hi - #s Ser Ile Phe Asp Arg                  500      - #           505      - #           510                  - - Tyr Asn Asp Ser Tyr Leu Arg Arg Ile Gly Th - #r Thr Ile Leu Ser Tyr              515          - #       520          - #       525                      - - Val Asn Met Val Cys Ala Thr Leu Arg Asn Se - #r Ile Pro Lys Ser Ile          530              - #   535              - #   540                          - - Val Tyr Cys Gln Val Arg Glu Ala Lys Arg Se - #r Leu Leu Asp His Phe      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Phe Thr Glu Leu Gly Lys Met Glu Thr Lys Ar - #g Leu Ser Ser Leu        Leu                                                                                             565  - #               570  - #               575             - - Asn Glu Asp Pro Ala Ile Met Glu Arg Arg Se - #r Ala Leu Ala Lys Arg                  580      - #           585      - #           590                  - - Leu Glu Leu Tyr Arg Ser Ala Gln Ala Glu Il - #e Asp Ala Val Ala Trp              595          - #       600          - #       605                      - - Ser Lys                                                                      610                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2211 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 175..2004                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GAATTCGGCA CGAGCGTCGT AAAAGCGAGT ATCCCGTTGG CAATTTGGCA TC -             #ACGTCCCT     60                                                                 - - TTTCAAACCA AGTTCCACAG ACACCAACAA CGCTTTAGCT CTCTCTTCTC TC -            #CGTCACCG    120                                                                 - - TCACCGGCGA TCTACATCTG AACCCGATCC AGCCAATAGA TCTCAGAAAT CC - #AA        ATG      177                                                                                      - #                  - #                  - #             Met                                                                                               - #                  - #                  - #                - - GAG AAC CTA ATC TCT TTG GTC AAC AAA ATC CA - #G AGA GCT TGC ACC        GCC      225                                                                    Glu Asn Leu Ile Ser Leu Val Asn Lys Ile Gl - #n Arg Ala Cys Thr Ala                        5    - #              10    - #              15                  - - TTA GGC GAC CAC GGA GAA AAC AGT GCA CTC CC - #C ACA CTA TGG GAC TCT          273                                                                       Leu Gly Asp His Gly Glu Asn Ser Ala Leu Pr - #o Thr Leu Trp Asp Ser                    20         - #         25         - #         30                      - - CTT CCC GCC ATC GCC GTC GTC GGA GGC CAG AG - #C TCA GGA AAG TCC TCC          321                                                                       Leu Pro Ala Ile Ala Val Val Gly Gly Gln Se - #r Ser Gly Lys Ser Ser                35             - #     40             - #     45                          - - GTC TTG GAG AGC GTT GTC GGC AAA GAT TTC TT - #A CCT CGT GGA TCA GGC          369                                                                       Val Leu Glu Ser Val Val Gly Lys Asp Phe Le - #u Pro Arg Gly Ser Gly            50                 - # 55                 - # 60                 - # 65       - - ATT GTT ACG CGA CGA CCT CTC GTG TTG CAG CT - #T CAC AAG ATT GAC GAG          417                                                                       Ile Val Thr Arg Arg Pro Leu Val Leu Gln Le - #u His Lys Ile Asp Glu                            70 - #                 75 - #                 80              - - GGA AGC AGG GAG TAC GCA GAG TTC CTC CAC CT - #C CCG AGG AAG AGG TTC          465                                                                       Gly Ser Arg Glu Tyr Ala Glu Phe Leu His Le - #u Pro Arg Lys Arg Phe                        85     - #             90     - #             95                  - - ACC GAT TTT GTT GCT GTG AGG AAG GAG ATT CA - #G GAC GAA ACT GAT AGA          513                                                                       Thr Asp Phe Val Ala Val Arg Lys Glu Ile Gl - #n Asp Glu Thr Asp Arg                   100          - #       105          - #       110                      - - GAG ACT GGA CGA ACC AAA CAA ATT TCG AGT GT - #T CCC ATT CAT CTT AGT          561                                                                       Glu Thr Gly Arg Thr Lys Gln Ile Ser Ser Va - #l Pro Ile His Leu Ser               115              - #   120              - #   125                          - - ATA TAC TCT CCT AAT GTT GTT AAC TTG ACG CT - #C ATT GAT CTT CCC GGC          609                                                                       Ile Tyr Ser Pro Asn Val Val Asn Leu Thr Le - #u Ile Asp Leu Pro Gly           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - CTT ACG AAA GTA GCT GTA GAG GGT CAA CCG GA - #T AGT ATT GTG AAA        GAC      657                                                                    Leu Thr Lys Val Ala Val Glu Gly Gln Pro As - #p Ser Ile Val Lys Asp                          150  - #               155  - #               160              - - ATT GAG GAT ATG GTT CGC TCC TAC ATT GAG AA - #G CCG AAC TGT ATA ATT          705                                                                       Ile Glu Asp Met Val Arg Ser Tyr Ile Glu Ly - #s Pro Asn Cys Ile Ile                       165      - #           170      - #           175                  - - TTG GCT ATT TCA CCA GCC AAT CAA GAT CTT GC - #A ACA TCC GAT GCA ATT          753                                                                       Leu Ala Ile Ser Pro Ala Asn Gln Asp Leu Al - #a Thr Ser Asp Ala Ile                   180          - #       185          - #       190                      - - AAA ATT TCC CGT GAA GTG GAC CCT ACT GGG GA - #T AGG ACC ATT GGA GTT          801                                                                       Lys Ile Ser Arg Glu Val Asp Pro Thr Gly As - #p Arg Thr Ile Gly Val               195              - #   200              - #   205                          - - TTG ACA AAG ATT GAT CTT ATG GAC AAG GGT AC - #T GAT GCT GTT GAT ATA          849                                                                       Leu Thr Lys Ile Asp Leu Met Asp Lys Gly Th - #r Asp Ala Val Asp Ile           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - TTG GAA GGA AGA GCA TAT AGG TTA AAG TTT CC - #C TGG ATT GGT GTT        GTG      897                                                                    Leu Glu Gly Arg Ala Tyr Arg Leu Lys Phe Pr - #o Trp Ile Gly Val Val                          230  - #               235  - #               240              - - AAT AGA TCA CAA CAA GAC ATA AAC AAG AAT GT - #T GAC ATG ATT GCT GCT          945                                                                       Asn Arg Ser Gln Gln Asp Ile Asn Lys Asn Va - #l Asp Met Ile Ala Ala                       245      - #           250      - #           255                  - - AGG CGT AGA GAA CGT GAG TAC TTT AAT AGT AC - #C CCT GAA TAT AAA CAC          993                                                                       Arg Arg Arg Glu Arg Glu Tyr Phe Asn Ser Th - #r Pro Glu Tyr Lys His                   260          - #       265          - #       270                      - - CTT GCA AAC AGA ATG GGT TCT GAA CAT CTG GC - #G AAG ATG CTC TCA AAG         1041                                                                       Leu Ala Asn Arg Met Gly Ser Glu His Leu Al - #a Lys Met Leu Ser Lys               275              - #   280              - #   285                          - - CAT TTG GAG ACA GTA ATC AAG TCC AAA ATT CC - #T GGC ATT CAA TCC CTA         1089                                                                       His Leu Glu Thr Val Ile Lys Ser Lys Ile Pr - #o Gly Ile Gln Ser Leu           290                 2 - #95                 3 - #00                 3 -      #05                                                                              - - ATT AAC AAA ACA ATT GCC GAA CTT GAA GCT GA - #A CTA ACT CGT TTA        GGA     1137                                                                    Ile Asn Lys Thr Ile Ala Glu Leu Glu Ala Gl - #u Leu Thr Arg Leu Gly                          310  - #               315  - #               320              - - AAA CCT GTT GCA GCT GAT GCT GGG GGA AAG TT - #G TAT GCT ATC ATG GAA         1185                                                                       Lys Pro Val Ala Ala Asp Ala Gly Gly Lys Le - #u Tyr Ala Ile Met Glu                       325      - #           330      - #           335                  - - ATA TGC CGC TCA TTT GAT CAA ATA TTT AAA GA - #C CAT CTT GAT GGC GTG         1233                                                                       Ile Cys Arg Ser Phe Asp Gln Ile Phe Lys As - #p His Leu Asp Gly Val                   340          - #       345          - #       350                      - - CGG CCT GGA GGT GAT AAA ATT TAT AAT GTC TT - #T GAC AAT CAG CTC CCT         1281                                                                       Arg Pro Gly Gly Asp Lys Ile Tyr Asn Val Ph - #e Asp Asn Gln Leu Pro               355              - #   360              - #   365                          - - GCT GCT TTA AAA AGG TTG CAG TTT GAT AAG CA - #G CTT TCA ATG GAA AAT         1329                                                                       Ala Ala Leu Lys Arg Leu Gln Phe Asp Lys Gl - #n Leu Ser Met Glu Asn           370                 3 - #75                 3 - #80                 3 -      #85                                                                              - - ATA AGG AAA CTT ATT ACA GAA GCT GAT GGG TA - #T CAG CCT CAT CTA        ATA     1377                                                                    Ile Arg Lys Leu Ile Thr Glu Ala Asp Gly Ty - #r Gln Pro His Leu Ile                          390  - #               395  - #               400              - - GCT CCA GAA CAA GGA TAC CGT CGC CTA ATT GA - #A TCT TCT CTA ATA ACT         1425                                                                       Ala Pro Glu Gln Gly Tyr Arg Arg Leu Ile Gl - #u Ser Ser Leu Ile Thr                       405      - #           410      - #           415                  - - ATT AGG GGC CCT GCT GAG TCA GCT GTT GAT GC - #G GTT CAC TCC CTG TTA         1473                                                                       Ile Arg Gly Pro Ala Glu Ser Ala Val Asp Al - #a Val His Ser Leu Leu                   420          - #       425          - #       430                      - - AAG GAC TTG GTT CAC AAA GCT ATG AGT GAG AC - #T TTG GAC TTG AAG CAG         1521                                                                       Lys Asp Leu Val His Lys Ala Met Ser Glu Th - #r Leu Asp Leu Lys Gln               435              - #   440              - #   445                          - - TAT CCT GGT CTC CGG GTT GAG GTT GGG GCT GC - #A TCT GTT GAT TCA CTC         1569                                                                       Tyr Pro Gly Leu Arg Val Glu Val Gly Ala Al - #a Ser Val Asp Ser Leu           450                 4 - #55                 4 - #60                 4 -      #65                                                                              - - GAA AGA ATG AGG GAT GAA AGC AAA AGA GCA AC - #A CTG CAG CTA GTT        GAT     1617                                                                    Glu Arg Met Arg Asp Glu Ser Lys Arg Ala Th - #r Leu Gln Leu Val Asp                          470  - #               475  - #               480              - - ATG GAG TGT GGC TAT CTG ACT GTT GAT TTC TT - #T CGG AAG CTT CCT CAA         1665                                                                       Met Glu Cys Gly Tyr Leu Thr Val Asp Phe Ph - #e Arg Lys Leu Pro Gln                       485      - #           490      - #           495                  - - GAT GTT GAT AAG GGT GGC AAT CCC ACA CAT TC - #A ATT TGT GAT AGA TAT         1713                                                                       Asp Val Asp Lys Gly Gly Asn Pro Thr His Se - #r Ile Cys Asp Arg Tyr                   500          - #       505          - #       510                      - - AAT GAT TCA TAT CTA AGG CGA ATT GGA ACC AC - #A ATT TTG TCA TAT GTC         1761                                                                       Asn Asp Ser Tyr Leu Arg Arg Ile Gly Thr Th - #r Ile Leu Ser Tyr Val               515              - #   520              - #   525                          - - AAT ATG GTC TGT GCT ACT CTG CGG CAT TCA AT - #T CCC AAG TCC ATC GTC         1809                                                                       Asn Met Val Cys Ala Thr Leu Arg His Ser Il - #e Pro Lys Ser Ile Val           530                 5 - #35                 5 - #40                 5 -      #45                                                                              - - TAT TGT CAA GTG CGG GAG GCA AAA CGA AGT CT - #A CTT GAT CAC TTT        TTT     1857                                                                    Tyr Cys Gln Val Arg Glu Ala Lys Arg Ser Le - #u Leu Asp His Phe Phe                          550  - #               555  - #               560              - - ACC GAG CTA GGC AAA ATG GAG ATC AAG CGT CT - #G TCC TCG TTA CTG AAT         1905                                                                       Thr Glu Leu Gly Lys Met Glu Ile Lys Arg Le - #u Ser Ser Leu Leu Asn                       565      - #           570      - #           575                  - - GAG GAT CCT GCA ATT ATG GAA CGA CGT AGT GC - #G CTC GCA AAG AGA CTA         1953                                                                       Glu Asp Pro Ala Ile Met Glu Arg Arg Ser Al - #a Leu Ala Lys Arg Leu                   580          - #       585          - #       590                      - - GAG TTA TAC CGG AGT GCA CAA GCT GAA ATA GA - #T GCA GTT GCT TGG TCT         2001                                                                       Glu Leu Tyr Arg Ser Ala Gln Ala Glu Ile As - #p Ala Val Ala Trp Ser               595              - #   600              - #   605                          - - AAG TAGAGATATG TATGTCAAAT CACGTTTATA CGAGAGCCAG CAGTGTCAT - #T              2054                                                                       Lys                                                                           610                                                                            - - ATCATTGTTC ACTATTTTCT TATTCATACT CATTTTTCAT TGTCATCTTA TT -             #CTGTTGCA   2114                                                                 - - TTTCCTCTTG AATAGATGAG ACGATTCTGA AAAAGGGAAA AAATGATTTT TT -            #GGGTTATA   2174                                                                 - - TATAATTGAG TGTCCCTATA TCTTTCATTT TTCAGTC      - #                      - #    2211                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 610 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Glu Asn Leu Ile Ser Leu Val Asn Lys Il - #e Gln Arg Ala Cys Thr        1               5 - #                 10 - #                 15              - - Ala Leu Gly Asp His Gly Glu Asn Ser Ala Le - #u Pro Thr Leu Trp Asp                   20     - #             25     - #             30                  - - Ser Leu Pro Ala Ile Ala Val Val Gly Gly Gl - #n Ser Ser Gly Lys Ser               35         - #         40         - #         45                      - - Ser Val Leu Glu Ser Val Val Gly Lys Asp Ph - #e Leu Pro Arg Gly Ser           50             - #     55             - #     60                          - - Gly Ile Val Thr Arg Arg Pro Leu Val Leu Gl - #n Leu His Lys Ile Asp       65                 - # 70                 - # 75                 - # 80       - - Glu Gly Ser Arg Glu Tyr Ala Glu Phe Leu Hi - #s Leu Pro Arg Lys Arg                       85 - #                 90 - #                 95              - - Phe Thr Asp Phe Val Ala Val Arg Lys Glu Il - #e Gln Asp Glu Thr Asp                  100      - #           105      - #           110                  - - Arg Glu Thr Gly Arg Thr Lys Gln Ile Ser Se - #r Val Pro Ile His Leu              115          - #       120          - #       125                      - - Ser Ile Tyr Ser Pro Asn Val Val Asn Leu Th - #r Leu Ile Asp Leu Pro          130              - #   135              - #   140                          - - Gly Leu Thr Lys Val Ala Val Glu Gly Gln Pr - #o Asp Ser Ile Val Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Ile Glu Asp Met Val Arg Ser Tyr Ile Gl - #u Lys Pro Asn Cys        Ile                                                                                             165  - #               170  - #               175             - - Ile Leu Ala Ile Ser Pro Ala Asn Gln Asp Le - #u Ala Thr Ser Asp Ala                  180      - #           185      - #           190                  - - Ile Lys Ile Ser Arg Glu Val Asp Pro Thr Gl - #y Asp Arg Thr Ile Gly              195          - #       200          - #       205                      - - Val Leu Thr Lys Ile Asp Leu Met Asp Lys Gl - #y Thr Asp Ala Val Asp          210              - #   215              - #   220                          - - Ile Leu Glu Gly Arg Ala Tyr Arg Leu Lys Ph - #e Pro Trp Ile Gly Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Asn Arg Ser Gln Gln Asp Ile Asn Lys As - #n Val Asp Met Ile        Ala                                                                                             245  - #               250  - #               255             - - Ala Arg Arg Arg Glu Arg Glu Tyr Phe Asn Se - #r Thr Pro Glu Tyr Lys                  260      - #           265      - #           270                  - - His Leu Ala Asn Arg Met Gly Ser Glu His Le - #u Ala Lys Met Leu Ser              275          - #       280          - #       285                      - - Lys His Leu Glu Thr Val Ile Lys Ser Lys Il - #e Pro Gly Ile Gln Ser          290              - #   295              - #   300                          - - Leu Ile Asn Lys Thr Ile Ala Glu Leu Glu Al - #a Glu Leu Thr Arg Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Lys Pro Val Ala Ala Asp Ala Gly Gly Ly - #s Leu Tyr Ala Ile        Met                                                                                             325  - #               330  - #               335             - - Glu Ile Cys Arg Ser Phe Asp Gln Ile Phe Ly - #s Asp His Leu Asp Gly                  340      - #           345      - #           350                  - - Val Arg Pro Gly Gly Asp Lys Ile Tyr Asn Va - #l Phe Asp Asn Gln Leu              355          - #       360          - #       365                      - - Pro Ala Ala Leu Lys Arg Leu Gln Phe Asp Ly - #s Gln Leu Ser Met Glu          370              - #   375              - #   380                          - - Asn Ile Arg Lys Leu Ile Thr Glu Ala Asp Gl - #y Tyr Gln Pro His Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ile Ala Pro Glu Gln Gly Tyr Arg Arg Leu Il - #e Glu Ser Ser Leu        Ile                                                                                             405  - #               410  - #               415             - - Thr Ile Arg Gly Pro Ala Glu Ser Ala Val As - #p Ala Val His Ser Leu                  420      - #           425      - #           430                  - - Leu Lys Asp Leu Val His Lys Ala Met Ser Gl - #u Thr Leu Asp Leu Lys              435          - #       440          - #       445                      - - Gln Tyr Pro Gly Leu Arg Val Glu Val Gly Al - #a Ala Ser Val Asp Ser          450              - #   455              - #   460                          - - Leu Glu Arg Met Arg Asp Glu Ser Lys Arg Al - #a Thr Leu Gln Leu Val      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asp Met Glu Cys Gly Tyr Leu Thr Val Asp Ph - #e Phe Arg Lys Leu        Pro                                                                                             485  - #               490  - #               495             - - Gln Asp Val Asp Lys Gly Gly Asn Pro Thr Hi - #s Ser Ile Cys Asp Arg                  500      - #           505      - #           510                  - - Tyr Asn Asp Ser Tyr Leu Arg Arg Ile Gly Th - #r Thr Ile Leu Ser Tyr              515          - #       520          - #       525                      - - Val Asn Met Val Cys Ala Thr Leu Arg His Se - #r Ile Pro Lys Ser Ile          530              - #   535              - #   540                          - - Val Tyr Cys Gln Val Arg Glu Ala Lys Arg Se - #r Leu Leu Asp His Phe      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Phe Thr Glu Leu Gly Lys Met Glu Ile Lys Ar - #g Leu Ser Ser Leu        Leu                                                                                             565  - #               570  - #               575             - - Asn Glu Asp Pro Ala Ile Met Glu Arg Arg Se - #r Ala Leu Ala Lys Arg                  580      - #           585      - #           590                  - - Leu Glu Leu Tyr Arg Ser Ala Gln Ala Glu Il - #e Asp Ala Val Ala Trp              595          - #       600          - #       605                      - - Ser Lys                                                                      610                                                                      __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide encoding aphragmoplastin protein having a molecular weight of about 65 to 70 kDaand at least one GTP binding domain, wherein said phragmoplastin proteinhas an amino acid sequence selected from the group consisting of theamino acid sequence of SEQ ID NO. 4 and the amino acid sequence of SEQID NO.
 6. 2. The isolated polynucleotide of claim 1, wherein theisolated polynucleotide encodes a phragmoplastin protein comprising theamino acid sequence of SEQ ID NO.
 4. 3. The isolated polynucleotide ofclaim 1, wherein the isolated polynucleotide encodes a phragmoplastinprotein comprising the amino acid sequence of SEQ ID NO.6.
 4. Theisolated polynucleotide of claim 1 wherein the polynucleotide comprisesa sequence selected from the group consisting of(a) the nucleotidesequence of the protein coding region of SEQ. ID. NO. 3; (b) thenucleotide sequence of the protein coding region of SEQ. ID. NO.5: and(c) a sequence which is complementary to the nucleotide sequence of (a)or (b).
 5. The isolated polynucleotide of claim 1 wherein thepolynucleotide is DNA.
 6. The isolated polynucleotide of claim 1 whereinthe polynucleotide encodes a chimeric phragmoplastin protein whichcomprises a phragmoplastin protein fused to a luminescent peptide tag.7. The isolated polynucleotide of claim 1 wherein the peptide tag is afluorescent protein.